PROJECT SUMMARY/ABSTRACT (NOTE: blue text indicates main resubmitted changes) This proposal addresses PQ5: How does mitochondrial heterogeneity influence tumorigenesis or progression? Mitochondria contain multiple copies of a non-nuclear genome (mtDNA), produce ATP and intermediate metabolites, support cell proliferation, but also have been shown in numerous studies to increase cancer risk, initiation, progression and impact responses to therapy when dysfunctional. Surprisingly, mitochondria translocate into stressed cells, including cancer cells, from the microenvironment. Mitochondria transfer into cancer cells can increase mtDNA heteroplasmy, but it is unknown how frequently translocated mitochondria integrate and the kinetics and extent of mtDNA expansion. It is also unknown how changes in gene expression, metabolism, growth, and structural alterations from transferred mitochondria promote tumorigenesis or cancer progression. Modeling transfer could improve quantitation and provide mechanistic insights that would be difficult to obtain in vivo. Fortunately, we invented a large cargo transfer device, a Biophotonic Laser Assisted cell Surgery Tool (BLAST), to enable quantitative transfer of mitochondria with specific mtDNA sequences into cancer or non-malignant cells. We will deploy this enabling technology to study our central hypothesis that cancer cells expand translocated mtDNA at rates that depend on mtDNA sequence and copy number, and that acquired mitochondria alter cell functions beyond energetics, including affecting cell rigidity to enhance metastatic potential and increase resistance to therapy. We will deploy BLAST to generate dozens to hundreds of mtDNA-nDNA hybrid cancer clones by transferring mitochondria with different WT or mutant mtDNA sequences and amounts into human cancer cells with or without endogenous mtDNA. We will quantify mtDNA expansion over time by three-independent approaches to determine sequence and copy number dependence for introduced mtDNA expansion or rejection. We will quantify the effects of mtDNA expansion on cancer-relevant respiration, signaling, growth, and proliferation by ?omics and novel quantitative phase microscopy approaches. Our proposal will improve our understanding of factors that contribute to tumor heterogeneity, aggressiveness, and resistance to therapy. Studies on how cancer cells adjust to translocated mitochondria opens opportunities for studies in cell-to-cell communications by organelle transfer and mitochondria rewiring of genome topology, gene expression, and changes in cancer cell proliferation and mechanical properties. This knowledge may lead to therapeutics that regulate mitochondria transfer to block cancer cell proliferation and impair biophysical changes that promote metastatic behavior.